Automated measurement of coagulation and fibrinolytic activation markers: Outcomes in coronavirus disease 2019 (COVID‐19) patients

Abstract Background Severe coronavirus disease 2019 (COVID‐19) is characterized by marked hypoxaemia and lung oedema, often accompanied by disordered blood coagulation and fibrinolytic systems, endothelial damage and intravascular fibrin deposition. Patients/Methods We present a retrospective observational study of 104 patients admitted to hospital with COVID‐19. Plasma samples were collected within 72 h of admission. In addition to routine coagulation and haematology testing, soluble thrombomodulin (sTM), thrombin‐antithrombin (TAT), tissue plasminogen activator‐plasminogen activator inhibitor 1 complex (tPAI‐C) and plasmin‐α2 antiplasmin complex (PIC) were performed by automated chemiluminescent enzyme immunoassays. Results Significantly higher levels of D‐dimer, TAT, sTM and tPAI‐C were observed in non‐survivors compared to survivors. To confirm which parameters were independent risk factors for mortality, multiple logistic regression was performed on D‐dimer, TAT. sTM, tPAI‐C and PIC data. Only increasing sTM was significantly associated with mortality, with an odds ratio of 1.065 for each 1.0 TU/mL increment (95% CI 1.025–1.115). Conclusions Of the haemostatic variables measured, sTM, which can be rapidly assayed, is the best independent predictor of mortality in patients hospitalized with COVID‐19, and this suggests that endothelial dysfunction plays an important role in disease progression.


| INTRODUCTION
Coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome caused by coronavirus-2 (SARS-CoV-2). Severe disease is characterized by marked hypoxaemia and lung oedema, often accompanied by disordered blood coagulation and fibrinolytic systems, endothelial damage and intravascular fibrin deposition. 1 Coagulation abnormalities are associated with poor prognosis. 2 Pulmonary immunothrombosis 1 and venous thromboembolic disease 3 are common in hospitalized patients with COVID-19. Despite this, overt disseminated intravascular coagulation, as defined by International Society on Thrombosis and Haemostasis criteria, 4 is uncommon in patients with COVID-19, 5,6 with little evidence of consumptive coagulopathy in most patients.
Increased D-dimer, with longer prothrombin time (PT) and activated partial thromboplastin time (APTT) on admission are associated with increased mortality. 2 Several authors have reported that increased levels of tissue plasminogen activator inhibitor-1 (PAI-1), [7][8][9] tissue plasminogen activator (tPA) 7 and soluble thrombomodulin (sTM) 7,9 are associated with increased mortality. However, most of the methods used were enzyme-linked immunoassays (ELISAs), which are not suitable for rapid testing. We hypothesized that rapid automated assays of coagulation and fibrinolytic activation markers may provide additional information on the coagulopathy associated with severe COVID-19.
We present a retrospective observational study in which rapid automated coagulation and fibrinolytic activation markers were analysed by chemiluminescent enzyme immunoassays (CLIEA), on plasma samples in addition to routine haematology testing, collected within 72 h of admission from patients admitted to hospital with COVID-19.

| MATERIALS AND METHODS
A total of 393 citrated plasma samples received by the coagulation laboratory at the Royal London Hospital (RLH) for routine analysis, were collected between 4 April 2020 and 31 December 2020 from patients with clinical details containing "COVID" or "corona." Due to the pressures of the pandemic, it was only possible to save a limited number of samples and samples were not collected from consecutive patients (over 15 000 patients with COVID have been admitted to Barts Health Hospitals during the pandemic).
All samples were centrifuged within 1 h of collection, and plasma was double-spun, separated and frozen below À70 C within 4 h. The samples were anonymized after recording the sex, age, ventilation status, SARS-Cov-2 PCR results, routine haematology results, date of admission and date of discharge or death. It was not possible to follow up patients after discharge. The saved plasma used does not fall under the auspices of the Human Tissue Act, 10

| RESULTS
The median age of the 104 patients from whom the samples were analysed was 58 years and 48% were female (Table 1) (Table 1). Longer PTs were observed in CPAP and ventilated than non-ventilated patients. This did not appear to be associated with abnormal liver function tests as otherwise reported by the laboratory. 29/104 samples (28%) had detectable anti-Xa levels (median 0.25 IU/dL, range 0.11-1.61 IU/mL). Ventilation status had no significant effect on APTT, fibrinogen, antithrombin, lymphocyte count, or platelet count.
TAT levels were significantly higher in ventilated patients than patients receiving CPAP or non-ventilated patients (Table 1) and were also significantly higher in patients receiving CPAP than nonventilated patients. Median sTM levels were higher in ventilated than the CPAP (2.5-fold higher) and non-ventilated patients (4-fold higher), although the difference only achieved statistical significance between MV and non-ventilated patients.
Overall 26.9% died including 40.0% of those on MV, 41.2% of those on CPAP and 12.0% of those not requiring advanced breathing support. Non-survivors were significantly older than surviving patients (Table 2), but no significant differences in sex were observed. Longer PTs and APTTs were observed in non-survivors but no significant differences in antithrombin or fibrinogen were seen. Non-survivors had significantly higher WBC and neutrophil counts, but lower lymphocyte counts, than survivors. There was no significant difference in platelet count between survivors and non-survivors.
Significantly higher levels of D-dimer, TAT, sTM and tPAI-C were observed in non-survivors. ROC analysis demonstrated that increased sTM levels had the highest discriminatory capacity of the test parameters studied. Patients with sTM above the reference interval (n = 38) were more likely to die than patients with normal levels (n = 61) with an odds ratio of 11.32 (95% CI 4.01-33.2, sensitivity 88.9% and specificity of 59.2%) for mortality. ROC analysis showed that a D-dimer cut off value of >0.8 mg/L had the best discriminatory function (sensitivity 92.6%, specificity 52.7%), which had an odds ratio of 7.52 (95% CI 2.19-24.81). Survivors with sTM above the reference interval on admission spent twice as long in hospital compared to those with normal levels (median 7 days vs. 15 days), although this was not statistically significant.
TAT and PIC were positively correlated with D-dimer levels (r = .64 and r = .62 respectively), whereas neither sTM nor tPAI-C demonstrated a significant relationship with D-dimer levels.
We analysed our data using PCA, a statistical pattern detection tool that reduces the dimensionality of a complex data set by means of a covariance matrix. This is used to create principal components, which are constructed as linear combinations of the initial variables in such a way that PCA1 explains the maximal amount of variance, while PCA2 accounts for the next highest variance. Thus PCA identifies clusters of variables that move together as groups. When applied to our data, PCA demonstrated that sTM and tPAI-C were clustered, and were distinct from D-dimer, TAT and PIC which were dependent on each other (Figure 1). To confirm which parameters were independent risk factors for mortality, multiple logistic regression was performed on D-dimer, TAT. sTM, tPAI-C and PIC data. Only increasing sTM was significantly associated with mortality, with an odds ratio of 1.065 for    The limitations of our study should be acknowledged. As the samples were anonymised, only limited clinical information was available on the patients. Due to the pressures of the pandemic on the coagulation laboratory, samples were not collected from consecutive patients.
Our data support growing evidence that endothelial dysfunction plays an important role in the pathophysiology of COVID-19, and is associated with poor outcomes. 5,16,17 While D-dimer may identify patients who will benefit from therapeutic-dose LMWH, 18 others have found D-dimer to be non-discriminatory. 19 It is thought that targeted interventions such as RAS inhibitors or statins may improve outcomes in patients with endothelial damage, by reducing NFκB (and hence tissue factor) expression and attenuating the effects of vascular endothelial growth factor. 16,20 Of the haemostatic variables measured, sTM, which can be rapidly assayed, is the best independent predictor of mortality in patients hospitalized with COVID-19, and this suggests that endothelial dysfunction plays an important role in disease progression. We believe that rapid measurement of haemostatic activation markers, particularly sTM, may be useful in guiding personalized medicine for patients with COVID-19.